This invention deals with a biochemical separation technique based on the principle of isoelectric focusing. This is an electrokinetic technique which takes advantage of the fact that many biochemical components, such as proteins, viruses, cells and cellular organelles, exhibit amphoteric qualities.
An amphoteric compound is one which has a number of charged, or potentially charged, acidic and basic moieties in its structure. The number of these acidic and basic moieties which will be charged at any time is dependent on the pH of the surrounding media, and there exists a certain pH at which the number of oppositely charged moieties will be exactly equal, making the net charge of the compound zero. This is called the isoelectric point, or pI. When an amphoteric compund is placed in a pH gradient and an electric potential is applied to it, it will migrate through the pH gradient until it reaches the point at which its net charge is zero. This technique of "focusing" an amphoteric compound at its pI has been in common laboratory use on an analytical scale for the last decade. Since different amphoteric compounds have different isoelectric points, a mixture of a large number of such compounds can be resolved into its component parts in one step, making this a powerful and popular technique.
A number of procedures and devices have been introduced to utilize the principle of isoelectric focusing in biochemical separations (for a complete discussion see P. G. Righetti and J. W. Drysdale, Laboratory Techniques in Biochemistry and Molecular Biology, T. S. Work and E. Work, eds., North Holland Publishing Company, Amsterdam, 1976, Vol. 5 and P. G. Righetti, ibid., 1983, Vol. 11).
One of the most common techniques involves the use of compounds known as carrier ampholytes to establish the pH gradient. These ampholytes are small, amphoteric buffer compounds which have their maximum buffering capacity at or near their pI. Therefore, when a mixture of them is placed in an electric field, they migrate to their isolelectric points with the various different types stacking perpendicular to the field, thereby establishing a pH gradient along the field.
There are number of disadvantages to the use of this procedure, most of which arise from the limitations of the ampholytes themselves. The first of these disadvantages involves a phenomenon known as cathodic drift. Briefly, the ampholytes tend to drift slowly in the direction of the cathode, causing the pH gradient to drift as well. This results in the focused proteins moving along with the gradient and losing resolution due to convection and mixing.
Also, the commonly available commercial ampholytes can produce only restricted pH gradients, typically with a range of 1-7 pH units. Since the resolution of the system is inversely proportional to the steepness of the gradient, the narrower the pH range the higher the resolution. It would therefore be desirable to have the ability to produce gradients with ranges significantly less than 1 pH unit.
The ampholytes can complex with the compounds being separated, causing anomalous bands at the pI of the complex rather than the true pI of the compound. It is therefore possible to get several different bands of the same compound, depending on the number of ampholytes with which it has complexed. This can seriously complicate separations in both analytical and preparative applications.
Often, especially in the case of preparative protein separations, the ampholytes must be removed from the purified components by subsequent steps because they interfere with several commonly-used procedures for the assay of peptides. This subsequent separation can be particularly difficult in the case of the short polypeptides, since their molecular weight and charge distribution is often very similar to that of the ampholytes which co-purify with them.
The ampholytes are relatively expensive, which tends to limit their practicality in preparative-scale applications.
Finally, once the pH gradient has been established there is no practical way of changing it during the run. This means that several different attempts are often necessary before the optimum conditions for the separation of a given compound are achieved. Even then, if there is more than one compound of interest to be purified from a mixture it is unlikely that the optimal conditions for one will also be optimal for the others. This often means that only one compound of a mixture can be adequately resolved by a given focusing run. Since a run can require up to three days to complete, this can become a serious drawback.
There have been numerous methods proposed for the formation of pH gradients without the use of carrier ampholytes. In one such method a step-gradient was produced by using membrane-bound compartments containing media at different pH values (A. J. P. Martin and F. Hampson, Journal of Chromatography, Vol. 159, p. 101, 1978). The membranes allowed passage of the material to be focused while maintaining the pH gradient. However, electroendosmotic flows carrying the buffer species along with the focusing material tended to disrupt the pH gradient, thereby lowering resolution. Also, the resolution was relatively low in any case due to the discontinuous nature of the gradient.
Another system involved the use of two equal and opposite streams of buffer at different pH values to establish a pH gradient in a convection-free zone bounded by the two streams (H. Rilbe, Journal of Chromatography, Vol. 159, p. 193, 1978 and U.S. Pat. No. 4,217,193). However, the pH gradient formed by this method is insufficiently stable for high-resolution focusing.
Yet another method has been proposed which involves the use of ion-selective membranes in conjunction with a chamber with non-parallel sides to produce a pH gradient (A. D. Hausfeld, U.S. Pat. No. 4,401,538). In this system, the non-parallel walls of the chamber cause the electric field lines generated by the anode and cathode means at either end of the chamber to assume different densities at different portions of the chamber. The focusing takes place in a secondary chamber having parallel walls which is nested inside the irregularly shaped chamber. These walls are constructed of ion-selective membranes which will pass ions of only one charge type. The ions of the solution will move at different rates in different portions of the chamber owing to the different field densities they experience. Those ions which are constrained inside the membrane compartment will not maintain equal distribution with regard to their counter-ions which can move freely through the membranes. This results in a charge imbalance which is balanced by the dissociation of water, creating a pH gradient.
The two main drawbacks to this procedure are: (1) it requires a power supply with very large current capacity, since most of the current flows through the outer chamber rather than through the focusing chamber. This necessitates special precautions to dissipate the additional heat caused by these large current flows. And (2), it shares the lack of flexibility in choice of gradients with the ampholyte technique (to change the gradient it is necessary to change the configuration of the chamber).
It is therefore an object of the present invention to provide an improved method for isoelectric focusing. A specific object of the present invention is to establish a pH gradient without the use of carrier ampholytes.
Another object of the present invention is to provide a method for establishing pH gradients with a range significantly less than 1 pH unit.
It is another object of the present invention to provide a means of altering the shape, range and median value of a pH gradient during the focusing run.
Still another object of the present invention is to provide a method of and apparatus for isoelectric focusing which will be compatible with high-capacity and continuous flow-through preparative applications.
It is also an object of the present invention to provide a method of and apparatus for isoelectric focusing in which the operator can select from a wide range of buffer solutions in which the focusing may take place.